Objective: To determine the construct validity, convergent validity, construct reliability and internal consistency of the Medical Student Stressor Questionnaire (MSSQ) among first-year medical students in Malaysia. Methods: A multicenter cross-sectional study was done on 375 medical students of four medical schools in Malaysia. The confirmatory factor analysis and reliability analysis were applied to measure construct validity, construct reliability and internal consistency of the MSSQ. These analyses were done using Predictive Analytics SoftWare (PASW) version 18 and Analysis of Moment Structure (AMOS) version 19. The Composite Reliability and Average Variance Extracted of the final constructs were calculated manually to determine construct reliability and convergent validity. Results: A total of 359 (95.7%) medical students responded to this study. The confirmatory factor analysis showed the six factor model with 20 items had a good fit with the latent constructs (X2 (df) = 258.02 (155), p < 0.001, RMR = 0.055, GFI = 0.933, AGFI = 0.910, NFI = 0.931, RFI = 0.916, IFI = 0.971, TLI = 0.965, CFI = 0.971, RMSEA = 0.043). The Cronbach’s alpha value of the MSSQ was 0.92. The Cronbach’s alpha values of the six constructs were more than 0.7. Composite Reliability and Average Variance Extracted values of the six constructs were more than 0.6 and 0.5 respectively indicating good construct reliability and adequate convergent validity. Conclusion: This study suggested that the six factor model with 20 items of the MSSQ had a good fit and shown good psychometric values. It is a valid and reliability measurement to identify stressors among medical students across institutions in Malaysia.